(M1030-01-04) How Do Extracellular Vesicles Enter Recipient Brain Endothelial Cells?

Purpose: Extracellular vesicles (EVs) are nano-sized bilayered membrane sacs that transfer cellular cargo in between cells(1-3). EVs transfer proteins, nucleic acids and lipids between donor and recipient cells (4. EVs are of two key types and they differ in size: the 100-1000 nm medium-to-large EVs (m/lEVs) that are also referred to as microvesicles and the 50-200 nm small EVs (sEVs) that are commonly referred to as exosomes (5). Their role in intercellular communication propelled the development of EVs as carriers for drug delivery. Owing to its natural origin EVs and low immunogenicity, EVs may show a superior performance as drug delivery carriers (6). Our laboratory has previously demonstrated that m/lEVs, but not sEVs, transfer their innate mitochondria to recipient brain endothelial cells (BECs). This mitochondrial transfer resulted in functional increases in ATP levels and mitochondrial function in the recipient BECs. We are currently developing m/lEVs as mitochondria delivery vehicles to BECs lining the BBB as a potential therapy for stroke (7-10). In this work, we seek to understand the mechanism by which m/lEVs vs. sEVs enter and the recipient BECs. While endocytosis is reported to be key mechanism via which cells internalize EVs (11, the entry pathway of EVs into BECs is currently not known. To study the cellular uptake mechanism, we individually blocked different routes of endocytosis using the respective pharmacological inhibitors and determined its effect on EV uptake into recipient BECs.

Methods: m/lEVs and sEVs were isolated from EV conditioned medium of hCMEC/D3 BECs using a differential ultracentrifugation method (6, 7, 10). We measured total EV protein content using a MicroBCA assay. We used dynamic light scattering (DLS) to measure EV particle size, dispersity index and zeta potential. We used western blotting to detect the presence of characteristic EV markers. EVs were labelled with calcein AM dye using our previously reported methods (6, 7, 10). Intracellular uptake of calcein-AM-EVs into recipient hCMEC/D3 cells was studied using fluorescence microscopy. Sucrose, genistein and 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) were used as the endocytosis inhibitors for clathrin-mediated endocytosis, clathrin-independent endocytosis and macropinocytosis pathways, respectively. Flow cytometry was used as a quantitative technique to study the effect of different inhibitors on the uptake of EVs into recipient BECs.

Results: sEVs and m/lEVs showed particle diameters of 126.3±7.8 nm and 251.4±3.2 nm, respectively, while both EV subpopulations showed a dispersity index of 0.3. Both EV subpopulations showed intracellular uptake within the recipient BECs as seen using z-stack fluorescence images. We noted that blocking macropinocytosis using EIPA showed the most significant reduction in m/lEV and sEV uptake into recipient BECs. Interestingly, we did not see an effect of blocking the caveolae pathway using genistein on the uptake of EVs.

Conclusion: We confirmed the intracellular uptake of EVs using the z-stack fluorescence microscopy and ruled out the possibility that EVs merely stick to the cell surface. Our results suggest that both EV subpopulations use macropinocytosis to enter the recipient BECs.

References: 1. Mulcahy LA, Pink RC, Carter DR. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles. 2014;3.
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3. Hong C-S, Funk S, Muller L, Boyiadzis M, Whiteside TL. Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. Journal of extracellular vesicles. 2016;5(1):29289.
4. Bonsergent E, Grisard E, Buchrieser J, Schwartz O, Thery C, Lavieu G. Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells. Nat Commun. 2021;12(1):1864.
5. Vader P, Mol EA, Pasterkamp G, Schiffelers RM. Extracellular vesicles for drug delivery. Advanced drug delivery reviews. 2016;106:148-56.
6. Dave KM, Zhao W, Hoover C, D'Souza A, D SM. Extracellular Vesicles Derived from a Human Brain Endothelial Cell Line Increase Cellular ATP Levels. AAPS PharmSciTech. 2021;22(1):18.
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8. D'Souza A, Burch A, Dave KM, Sreeram A, Reynolds MJ, Dobbins DX, Kamte YS, Zhao W, Sabatelle C, Joy GM. Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells. Journal of Controlled Release. 2021;338:505-26.
9. Manickam DS. Delivery of mitochondria via extracellular vesicles–A new horizon in drug delivery. Journal of Controlled Release. 2022;343:400-7.
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11. Costa Verdera H, Gitz-Francois JJ, Schiffelers RM, Vader P. Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis. J Control Release. 2017;266:100-8.

Fig. 2. Uptake of calcein-EVs into the recipient hCMEC/D3 cells using fluorescence microscopy. hCMEC/D3 BECs were cultured in 24-well plates until 80% confluency in a humidified incubator. Cells were then incubated with the indicated amounts of Calcein AM labelled-EVs in complete growth medium for 24 h. Post-incubation, the cells were washed, labelled with Hoechst stain and incubated with phenol-red-free growth medium (a, b, c). Intracellular EV signals were observed under an Olympus IX 73 epifluorescent inverted microscope using GFP channel (green puncta) at 20x magnification. Intracellular Hoechst labelled nucleus signals were observed using DAPI channel (false coloured as red signals) at 20x magnification (d and e). Scale bar: 50 µm.



Fig. 1- Scheme representing the different endocytosis pathways.



Fig. 3. Effect of inhibiting endocytosis pathways on EV uptake in the recipient BECs. Cells were pre-incubated for 0.5 h with the respective endocytosis inhibitors and then were treated for 4 h with fresh endocytosis inhibitors along with calcein-labelled m/lEVs and sEVs. % EV uptake was normalized to the untreated cells after analysing cells using an Attune flow cytometer. Dunnett's multiple comparisons test were used for comparative analysis using one-way ANOVA. Data are presented as mean±SD (n=3). ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001